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Investigating Nanoscale Drug Delivery Systems With Nano-IR Analysis

Various forms of nano-particles are being utilized as part of nanoscale drug delivery systems. Most delivery systems consist of core and shell components. Sometimes the shell is used as protective encapsulation and sometimes it is part of an active chemical. While the effectiveness of the drug delivery systems can be tested after the manufacturing process is completed, a technique that can validate each manufacturing process with molecular fingerprinting of individual nano-particle should be tremendously beneficial for quality control. The current array of molecular analytical instrumentation is not well suited to study the nanoscale drug delivery systems at the individual nano-particle due to the size of the nano-particles (generally ≤100 nm).

IR PiFM is an ideal tool to monitor these nano-particles. Except for metal cores, IR PiFM can provide molecular information on most of the important components such as ligands, drugs, and other chemicals that comprise the system. Figure 1 shows a model core-shell system composed of two polymeric materials, hydrophobic polystyrene core and hydrophilic polymethacrylate shell. Topography and phase from AFM reveal the spherical particles with no details on whether the core-shell structure is achieved. FTIR measurement should reveal the presence of both polymers, albeit with no details on phase distribution. When imaged with IR PiFM at 1493 cm−1 and 1720 cm−1, hydrophobic polystyrene core components and hydrophilic polymethacrylate shell are respectively mapped clearly. The cross-sectional analysis (shown in the far-right images) show that the shell thickness is about 9 nm while the core is about 18 nm in diameter. PiFM can image the core through the thin shell layer since PiFM measures signals down to ~ 15 nm in depth. The shell component appears as a donut since the tip integrates the signal underneath the tip down to about 15 nm; when the tip is at the edge of the particle, tip integrates the signal from more polymethacrylate molecules.

AFM topography with PiFM images and 3d visualization
Figure 1. Visualization of core-shell block copolymer by AFM (topography and phase images on the left) and PiFM. PiFM images at 1720 cm−1 and 1493 cm−1 are selectively mapping the hydrophilic polymethacrylate shell and hydrophobic polystyrene core, respectively. The cross-sectional measurements (images on the far right) show that the shell thickness is about 9 nm and the core diameter is about 18 nm.

In the next example (Figure 2), we map not only the ligand on nano-particles but also contaminants that are produced during the functionalization process of magnetic iron oxide NPs (MNPs). MNPs, especially magnetite (Fe3O4) NPs, have gained considerable attention due to their many applications in human diagnostics including MRI contrast enhancement. To change the surface of magnetite NPs from hydrophobic to hydrophilic for stable monodispersions in aqueous phases, the ligand from oleic acid (the initial surfactant of the core MNP) is exchanged for a multidentate dopamine-based tertiary amine ligand; the processes to synthesize the tertiary amine ligand bearing a dopamine-containing block copolymer and the exchanging of ligands are shown in Figure 2a and 2b, respectively. The TEM image of the NPs reveal the core NP to be ~ 5 – 7 nm in size (Figure 2c). For IR PiFM measurements, the functionalized NPs are prepared on a Si substrate via spin-coating. AFM topography (Figure 2d) shows a uniform distribution of the functionalized NPs along with a few larger globules, considered to be a contaminant. PiF-IR spectra on the contaminant and the MNPs are shown in Figure 2h along with the FTIR spectrum of the bulk DOPA-tertiary amine-MNPs (black dashed line) and a FTIR spectrum of oleic acid (red dotted line). The agreement between PiF-IR spectrum for the contaminant and a FTIR spectrum for oleic acid indicate that the contaminant arises from the residual oleic acid. The PiFM images at 1260 cm−1 and 1040 cm−1, both of which are vibrational bands associated with DOPA-tertiary amine-MNPs clearly highlight individual MNPs with donut-like appearance, like the shell polymethacrylate molecules in Figure 1. The PiFM image at 1120 cm−1 (IR band from oleic acid) highlights the contaminant particles; the donut-like appearance suggests that the contaminant particles also have core-shell structure with the oleic acid comprising the shell component.

Figure 2. PiFM and PiF-IR analysis of DOPA-tertiary amine-MNPs. (a) synthesis of the tertiary amine ligand bearing a dopamine-containing block copolymer; (b) exchange of the ligand from oleic acid for a multidentate dopamine-based tertiary amine ligand; (c) TEM image; (d) AFM topography; (e) – (g) PiFM images; and (h) PiF-IR spectra of MNP (black) and contaminant (red) with FTIR spectra for MNP (black dashed) and oleic acid (red dotted).

As an example of imaging an active pharmaceutical ingredient (API), we look at the miscibility and morphology of poly(ε-caprolactone) (PCL) and its blends with L-ascorbic acid (AA). PCL is a linear aliphatic polyester that is a biocompatible, non-toxic, and biodegradable polymer and has been used in various pharmaceutical and biomedical applications. By combining PCL with AA, several benefits are realized: (1) improved biodegradation rate; and (2) increased hydrophilicity of the biopolymer. Controlling the morphology and crystallization of polymers and composites is crucial because it is directly related to the final properties of the material. Figure 3 shows topography, PiFM image at 1725 cm−1 (highlighting PCL), two combined PiFM images, and PiF-IR spectra (color-coded from designated points in the topography image) along with bulk FTIR spectra for a sample with 5 wt% of AA concentration. From topography image and the associated PiF-IR, we can see that the PCL/AA blend is immiscible, and AA has crystallized into micrometric crystals. The PiF-IR spectra from the two PCL regions (one in the center of the crystal clusters (dark green) and another from the matrix region (teal)) are virtually identical to each other except for a minor difference in the C=O bond around 1725 cm−1 and agree well with the bulk FTIR; the two intensity dips in PiF-IR around 1180 cm−1 and 1250 cm−1 are artifacts due to a defective laser. The three PiF-IR spectra tell a different story as each PiF-IR spectrum produces mostly the same peaks with each other and bulk FTIR but with different relative intensities. PiFM images show the immiscibility clearly. PiFM image at 1725 cm−1 only highlights PCL molecules where AA crystals are absent. As was hinted by the spectra, we can see from the PiFM image that there are at least three levels of C=O bond intensity. PiFM images at 1666, 1119, and 821 cm−1 are false colored as blue, green, and red, respectively and combined to highlight only the AA crystallites (bottom left image). Since the excitation laser is p-polarized (electric field along the tip axis), we believe the different relative intensities arise from the different crystalline face that is exposed. The bottom middle image replaces the red map with the PCL band to clearly show the immiscibility. Given the richness of the peaks both in FTIR and PiF-IR spectra, the region in the red dotted rectangle is shown magnified in Figure 4. In the FTIR spectrum, we can see two clear doublets at locations A and B. However, in the PiF-IR spectra, different locations contribute differently to the doublets; at A, PiF-IR2 and PiF-IR3 contribute almost purely to the peak at the lower and the higher wavenumber respectively while PiF-IR1 shows a doublet, albeit not equally as in FTIR; at B, PiF-IR1 and PiF-IR2 contribute exclusively to the lower wavenumber while PiF-IR3 contributes exclusively to the higher wavenumber. IR PiFM’s capability to detect local chemical states with sub-10 nm spatial resolution should give chemists a powerful tool to understand chemical processes involved in the manufacturing and quality control of nanoscale drug delivery systems and the drug-body interactions.

Figure 3. Topography, PiFM image at 1725 cm−1 (highlighting PCL), two combined PiFM images, and PiF-IR spectra (color-coded from designated points in the topography image) along with bulk FTIR spectra for a PCL/AA blend sample with 5 wt% of AA concentration.
Figure 4. Magnified view of the spectra shown in red dotted rectangle in Figure 3 shows how different nanoscale PiF-IR spectra contribute to the doublets (marked A and B) observed in bulk FTIR spectrum.

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